Primary reactive oxygen species (ROS), such as superoxide radicals, hydrogen peroxide, hydroxyl radicals, and ortho-quinone derivatives of catecholamines, exert their cellular effects by modifying DNA, lipids, and proteins to form secondary electrophiles. Examples of such secondary electrophiles include hydroxyalkenals, nucleotide propenals, and hydroxyperoxy fatty acyl chains. Secondary electrophiles are implicated in cellular dysfunction either because they are no longer able to participate in normal cellular activity or because they serve as electron acceptors in oxidative chain reactions that result in the modification of other essential cellular components. Damage caused by primary and secondary ROS contributes to the pathogenesis of several acute human diseases. ROS likely participate in the central nervous system damage caused by neuronal ischemia during stroke, post-cardiopulmonary bypass syndrome, brain trauma, and status epilepticus, as well as the cardiac damage induced during ischemic heart disease and renal damage induced by ischemia and toxins. ROS also likely participate in chronic human diseases such as the destruction of the islets of Langerhans of the endocrine pancreas in Diabetes Mellitus, the destruction of neurons in Parkinson's disease, and other chronic neurodegenerative disorders.
One way that cells handle the deleterious effects of ROS is via the preconditioning response, an adaptation whereby cells are rendered resistant to injury by prior exposure to smaller doses of the same ROS-inducing stress, which threatens to cause the injury in question. Thus, an ideal therapeutic strategy for the prevention and/or treatment of diseases involving an oxidative stress would involve stimulation of the preconditioning response without causing cellular injury. However, identification of potentially useful therapeutic agents has been hampered by the fact that compounds that cause the preconditioning response generally also cause stress to the cell.
Recently, the signaling pathways involved in the cellular response to oxidative stress have been elucidated (see FIG. 1). For example, the accumulation of malfolded proteins in the endoplasmic reticulum (ER stress) leads to accumulation of ROS. ER stress activates the protein kinase PERK, which in turn phosphorylates the translation initiation factor eIF2 on its alpha subunit [Harding, H., Zhang, Y., and Ron, D. (1999). Translation and protein folding are coupled by an endoplasmic reticulum resident kinase. Nature 397, 271–274]. A different eIF2α kinase, GCN2, phosphorylates eIF2α in response to nutritional stress [Harding, H., Novoa, I., Zhang, Y., Zeng, H., Schapira, M., and Ron, D. (2000). Regulated translation initiation controls stress-induced gene expression in mammalian cells. Mol. Cell 6, 1099–1108]. eIF2α phosphorylation leads to marked reduction in protein biosynthesis [Harding, H., Zhang, Y., Bertolotti, A., Zeng, H. and Ron, D. (2000). Perk is essential for translational regulation and cell survival during the unfolded protein response. Mol. Cell 5, 897–904] and to the expression of the transcription factor ATF4, which then activates stress response genes in a signaling pathway termed the Integrated Stress Response (ISR) [Harding, H., Novoa, I., Zhang, Y., Zeng, H., Schapira, M., and Ron, D. (2000). Regulated translation initiation controls stress-induced gene expression in mammalian cells. Mol. Cell 6, 1099–1108].
This activated Integrated Stress Response pathway is down-regulated by the activity of a phosphatase holoenzyme that dephosphorylates eIF2α on serine 51 (in yeast eIF2α, corresponding to residue 52 in rodents or humans). The phosphatase holoenzyme consists of the catalytic subunit of protein phosphatase 1 (PP1c) and GADD34, an eIF2α-specific regulatory subunit of the phosphatase [Novoa, I.; Zeng, H., Harding, H., and Ron, D. (2001). Feedback inhibition of the unfolded protein response by GADD34-mediated dephosphorylation of eIF2α. J. Cell Biol., 153, 1011–1022].
The expression of Integrated Stress Response target genes promotes resistance to both the stress of malfolded proteins in the endoplasmic reticulum and to the consequences of ROS accumulation. Methods to monitor activation of the Integrated Stress Response provide for effective screens to identify test substances capable of promoting preconditioning by activating the pathway in cells not subject to stress. Specifically, these screening methods to assess activation of the ISR provide the advantage of the identification of test substances, which activate the pathway, yet do not cause cell stress.
Substances that inhibit the eIF2α-specific phosphatase could promote the accumulation of phosphorylated eIF2α in cells not subject to stress, and thereby activate a protective ISR response without subjecting the cell to stress. Such substances would therefore be useful in the prevention and/or treatment of diseases involving an oxidative stress. GADD34 is limited in its use as a therapeutic target in this scheme as it is only expressed following stress-induced activation of eIF2α kinases, and not in cells not subject to stress. Therefore, inhibition of GADD34 is unlikely to provide a protective ISR response against oxidative stress in cells not subject to stress.
This invention involves the discovery of a GADD34-related regulatory subunit of PP1c, hereinafter referred to as GADD34-Like or GADD34L. Inhibition of GADD34L activity in cells not subject to stress leads to increased phosphorylation of eIF2α and to activation of the ISR. Therefore, GADD34L represents a useful therapeutic target for the promotion of preconditioning to prevent and/or treat diseases involving an oxidative stress. Furthermore, screening for test substances that are inhibitors of GADD34L activity provides the advantage of identifying therapeutic agents to prevent and/or treat diseases involving oxidative stress by activation of the ISR pathway, that do not provide cells stress.